Method of increasing feed utilization

ABSTRACT

Antibiotic A-28086 complex, comprising microbiologically active, structurally related factors A, B, and D, produced by submerged aerobic fermentation of Streptomyces aureofaciens NRRL 5758. Individual factors A, B, and D are separated and isolated by chromatography. The A-28086 antibiotics are antibacterial, antifungal, antiviral, anti-PPLO, anticoccidial, insecticidal and acaricidal agents and also increase feed-utilization efficiency in ruminants.

CROSS-REFERENCE TO RELATED APPLICATION This is a division, of copendingapplication Ser. No. 569,740 filed Apr. 21, 1975, which in turn is acontinuation-in-part of application Ser. No. 477,954, filed June 10,1974, now abandoned. BACKGROUND OF THE INVENTION

1. Field of the Invention

Although many antibacterial agents are known today, there continues tobe a demand for new, improved antibiotics. One problem in currentantibiotic therapy is the fact that antibiotics differ in theireffectiveness against pathogenic organisms. Another problem is thedevelopment of organism strains which are resistant to standardantibiotics. Yet another problem is the fact that individual patientsoften suffer serious reactions to specific antibiotics, due tohypersensitivity and/or to toxic effects. Because of these problems incurrent therapy, new antibiotics continue to be in demand.

In addition to demands for new antibiotics which are useful in treatingdiseases in humans, improved antibiotics are also needed in theveterinary field. In one important aspect, improved antibiotics areneeded to promote growth in poultry and in livestock. Growth promotionis achieved, for example, by reducing disease and by increasingfeed-utilization efficiency.

One well-known disease of economic importance to veterinary science,more specifically to the poultry industry, is the protozoan diseasecoccidiosis. Coccidiosis results from infection by one or more speciesof Eimeria or Isospora (for a summary, see Lund and Farr in "Diseases ofPoultry," 5th ed, Biester and Schwarte, Eds., Iowa State UniversityPress, Ames, Ia., 1965, pp 1056-1096). In view of the great economiclosses due to coccidiosis and the disadvantages of some knownanticoccidial agents, the search for better anticoccidial agentscontinues.

Enteritis is another disease which can cause severe economic losses tolivestock producers. Enteritis occurs in chickens, swine, cattle andsheep and is attributed mainly to anaerobic bacteria, particularlyClostridium perfringens, and viruses. Enterotoxemia in ruminants, anexample of which is "overeating disease" in sheep, is a condition causedby C. perfringens infection.

Promotion of growth in ruminants, such as cattle, is anothereconomically desirable objective of veterinary science. Of particularinterest is growth promotion achieved by increasing feed-utilizationefficiency. The mechanism for utilization of the major nutritive portion(carbohydrates) of ruminant feeds is well known. Microorganisms in therumen of the animal degrade carbohydrates to produce monosaccharides andthen convert these monosaccharides to pyruvate compounds. Pyruvates aremetabolized by microbiological processes to form acetates, butyrates orpropionates, collectively known as volatile fatty acids (VFA). For amore detailed discussion, see Leng in "Physiology of Digestion andMetabolism in the Ruminant," Phillipson et al., Eds., Oriel Press,Newcastle-upon-Tyne, England, 1970, pp 408-410.

The relative efficiency of VFA utilization is discussed by McCullough inFeedstuffs, June 19, 1971, page 19; Eskeland et al. in J. An. Sci. 33,282 (1971); and Church et al. in "Digestive Physiology and Nutrition ofRuminants," Vol. 2, 1971, pp 622 and 625. Although acetates andbutyrates are utilized, propionates are utilized with greaterefficiency. Furthermore, when too little propionate is available,animals may develop ketosis. A beneficial compound, therefore,stimulates animals to produce a higher proportion of propionates fromcarbohydrates, thereby increasing carbohydrate-utilization efficiencyand also reducing the incidence of ketosis.

2. The Prior Art

Antibiotic A-28086 factors A, B, and D are new members of a group ofpolyether antibiotics. Examples of members of this group includemonensin (U.S. Pat. No. 3,501,568); dianemycin [R. L. Hamill, M. M.Hoehn, G. E. Pittenger, J. Chamberlin, and M. Gorman, J. Antibiotics 22,161 (1969)]; nigericin [L. K. Steinrauf, Mary Pinkerton, and J. W.Chamberlin, Biochem. Biophys. Res. Comm. 33 29 (1968)]; and salinomycin[Japan patent publication 47-25392, dated Oct. 20, 1972, application No.19620/1971; Derwent No. 76960T, U.S. Pat. No. 3,857,948; and H. Kinashi,N. Otake, H. Yonehara, S. Sato and Y. Saito, Tetrahedron Lett. 49,4955-4958 (1973).

SUMMARY OF THE INVENTION

This invention relates to antibiotic substances. In particular, itrelates to an antibiotic complex comprising at least three individualfactors. The complex comprising individual factors A, B, and D isproduced by culturing a hitherto undescribed strain of the organismStreptomyces aureofaciens Duggar, NRRL 5758.

The term "antibiotic complex" as used in the fermentation art and inthis specification does not refer to a chemical complex, but to amixture of co-produced individual antibiotic factors. As will berecognized by those familiar with antibiotic production by fermentation,the ratio of individual factors produced in an antibiotic complex willvary, depending on the fermentation conditions used.

The antibiotic substances in this invention are arbitrarily designatedherein as A-28086 antibiotics. The two individual antibiotics of thepresent invention are designated antibiotic A-28086 factors A and B. TheC₂ -C₆ -acyl ester derivatives of A-28086 factor A and thephysiologicallyacceptable salts of said ester derivatives and of A-28086factors A and B are also part of this invention. The A-28086 antibioticcomplex also contains individual factor D which is the subject of aseparate invention. To simplify discussions of utility, the term"A-28086 compound" is used and refers to individual factors A and B, thespecified acyl ester derivatives of factor A orphysiologically-acceptable salts as above defined. A-28086 factor D hasan activity pattern similar to that of A-28086 factors A and B.

The A-28086 antibiotic complex is produced by culturing the novel strainof Streptomyces aureofaciens NRRL 5758 under submerged aerobicfermentation conditions until a substantial level of antibiotic activityis produced. The A-28086 antibiotic complex can also be produced byculturing yet another novel strain of Streptomyces aureofaciens, NRRL8092. When produced by either S. aureofaciens NRRL 5758 or by S.aureofaciens NRRL 8092, the A-28086 antibiotic complex is extracted fromthe fermentation broth and from the mycelium with polar organicsolvents. The extracted antibiotic mixture is separated by concentratingthe solvents, adding the concentrates to excesses of petroleum ether toprecipitate impurities, filtering, and evaporating the filtrates toobtain the A-28086 antibiotic mixture. The antibiotic mixture is furtherpurified and separated into individual factors by column chromatography.

The A-28086 compounds of this invention inhibit the growth of organismswhich are pathogenic to animal and plant life. In one aspect of thisinhibitory activity, the A-28086 compounds are anticoccidial agents. Inaddition, the A-28086 compounds are antibacterial, antifungal,antiviral, anti-PPLO, insecticidal and acaricidal agents and increasefeed-utilization efficiency in ruminants.

DESCRIPTION OF THE DRAWINGS

The following infrared absorption spectra in chloroform are presented inthe drawings:

FIG. 1 - Antibiotic A-28086 factor A

FIG. 2 - Antibiotic A-28086 factor B

FIG. 3 - Antibiotic A-28086 factor A acetyl ester derivative

FIG. 4 - Antibiotic A-28086 factor A propionyl ester derivative

FIG. 5 - Antibiotic A-28086 factor A butyryl ester derivative

FIG. 6 - Antibiotic A-28086 factor A valeryl ester derivative

FIG. 7 - Antibiotic A-28086 factor A caproyl ester derivative

DETAILED DESCRIPTION OF THE INVENTION

The A-28086 factors of this invention are structurally related to eachother. At least four antibiotic factors are coproduced during thefermentation and are obtained as a mixture. The factors are separatedfrom each other, and factors A, B, and D are isolated as individualcompounds as hereinafter described. The mixture of A-28086 factors issoluble in most organic solvents, but is insoluble in water.

The following paragraphs describe the physical and spectral propertiesof A-28086 factors A and B, the two factors of the present invention.

Antibiotic A-28086 factor A crystallizes from acetone-water. A-28086factor A melts at about 98°-100° C., resolidifies and remelts at about195°-200° C. Elemental analysis of factor A gave the following averagepercentage composition: carbon, 66.69 percent; hydrogen, 9.85 percent;oxygen, 23.10 percent. The empirical formula proposed for factor A isC₄₃ H₇₂ O₁₁.

Factor A has an apparent molecular weight of 764 as determined by massspectrometry.

The infrared spectrum of factor A in chloroform is shown in FIG. 1 ofthe accompanying drawings. The following absorption maxima are observed:2.85, 3.34, 5.83, 6.82, 7.22, 7.53 (weak), 7.78 (weak), 8.75 (strong),8.95 (strong), 9.15, 9.50 (strong), 9.55 (strong), 9.60, 9.85, 10.15,10.45, and 10.70 (weak) microns.

The ultraviolet spectrum of factor A in ethanol shows only endabsorption below 220 mμ.

The nuclear magnetic resonance spectrum of A-28086 factor A indeuterochloroform showed the following characteristics: δ 6.01, 4.21,4.11, 3.99, 3.89, 3.80, 3.67, 3.65, 3.57, 3.55, 2.83, 2.76, 2.74, 2.68,2.66, 2.58, 2.56, 2.30, 2.22, 2.17, 2.10, 2.05, 1.96, 1.90, 1.85, 1.70,1.62, 1.60, 1.47, 1.39, 1.31, 1.25, 1.18, 0.95, 0.93, 0.90, 0.88, 0.85,0.77, 0.75, 0.73, 0.68, and 0.66 ppm.

Antibiotic A-28086 factor A, crystallized from acetone-water, has thefollowing characteristic X-ray powder diffraction pattern (Cu⁺⁺radiation, 1.5405λ, nickel filter, d = interplanar spacing inangstroms):

    ______________________________________                                                       Relative                                                       d              Intensity                                                      ______________________________________                                        12.00          100                                                            10.10          50                                                             9.25           90                                                             8.00           40                                                             7.50           15                                                             6.92           90                                                             6.40           40                                                             5.98           05                                                             5.68           15                                                             5.20           40                                                             4.98           40                                                             4.62           40                                                             4.21           20                                                             3.48           10                                                             ______________________________________                                    

The specific rotation of antibiotic A-28086 factor A is -54° (c=0.2,methanol), when determined at a temperature of 25° C. This specificrotation is an average value, based on several determinations.

Electrometric titration of factor A in 80% aqueous dimethylformamideindicated the presence of a titratable group with a pK_(a) value of 7.9.

Antibiotic A-28086 factor A is soluble in a variety of organic solventssuch as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethylacetate, chloroform, acetone, and benzene; but is only slightly solublein nonpolar organic solvents such as hexane; and is insoluble in water.

Antibiotic A-28086 factor A has an acid function capable of formingsalts and ester derivatives and at least one hydroxyl group capable ofesterification.

Based on the physical characteristics hereinabove recited, a structurefor antibiotic A-28086 factor A can be proposed. Since the structuredetermination is merely postulated, however, it is to be understood thatthe structure presented herein represents merely a working hypothesis.The tentative structure for A-28086 factor A is shown in Formula I:##STR1##

Antibiotic A-28086 factor B is a white crystalline compound (fromacetone-water) which has a melting point of about 150°-153° C.

As determined by high-resolution mass spectrometry, factor B has anapparent molecular weight of 762 and a proposed empirical formula of C₄₃H₇₀ O₁₁.

The infrared spectrum of factor B in chloroform is shown in FIG. 2 ofthe accompanying drawings. The following absorption maxima are observed:2.82, 3.30, 5.77, 5.85, 6.80, 7.20, 7.50 (weak), 7.72 (weak), 7.80(weak), 8.57 (strong), 8.68, 8.90 (strong), 9.10, 9.50, 9.83 (strong),9.90, 10.10, 10.17 (strong), 10.43 (weak), 10.80 (weak), 11.20 (weak),11.35 (weak), 11.73 (weak), and 12.03 (weak) microns.

The ultraviolet spectrum of factor B in ethanol shows an absorptionmaximum at 220 mμ (E_(1cm) ^(1%) = 137.5; ε=10,477).

The nuclear magnetic resonance spectrum of A-28086 factor B indeuterochloroform showed the following characteristics: δ 7.20, 7.09,6.26, 6.15, 4.19, 4.12, 4.05, 3.95, 3.89. 3.78, 3.62, 3.59, 3.52, 3.48,2.81, 2.73, 2.63, 2.54, 2.52, 1.99, 1.91, 1.84, 1.71, 1.67, 1.64, 1.55,1.43, 1.33, 1.18, 1.11, 0.96, 0.94, 0.90, 0.87, 0.84, 0.77, 0.74, and0.68 ppm.

Antibiotic A-28086 factor B is soluble in a variety of organic solventssuch as, for example, methanol, ethanol, dimethylformamide, dimethylsulfoxide, ethyl acetate, chloroform, acetone and benzene; but is onlyslightly soluble in nonpolar organic solvents such as hexane; and isinsoluble in water.

Although the chemical structure of A-28086 antibiotic factor B has notbeen elucidated, the physicalchemical data thus far available indicatethat factor B has a single carboxylic acid moiety, two ketone moieties,and one or more hydroxyl moieties.

Antibiotic A-28086 factor D, when produced by S. aureofaciens NRRL 5758,is a minor factor. When produced by S. aureofaciens NRRL 8092, however,A-28086 factor D is present in amounts up to 10% of the recoveredantibiotic activity. Antibiotic A-28086 factor D is the subject of acopending application of Nakatsukasa and Hamill, titled ANTIBIOTICA-28086 FACTOR D AND PROCESS FOR PRODUCTION THEREOF, Ser. No. 569,719,filed this even date herewith.

The following paragraphs describe the physical and spectral propertiesof antibiotic A-28086 factor D.

Antibiotic A-28086 factor D is a white crystalline material (fromwater-acetone) with a melting point of about 96°-98° C. A-28086 factor Dhas an apparent molecular weight of 778, as determined byhigh-resolution mass spectrometry.

The elemental composition of the peak in the mass spectrum of the sodiumsalt of A-28086 factor D was observed to be 800.5050 (Calcd for C₄₄ H₇₃O₁₁ Na = 800.5050). In the mass spectrum of A-28086 factor D free acid,a small peak at 778 and a larger peak at 760.5177 (Calcd for C₄₄ H₇₂ O₁₀= 760.5125) were observed. The m/e 760 in the mass spectrum of the freeacid results from the loss of water from the molecular ion. Themolecular-ion composition of A-28086 factor D free acid is, therefore,C₄₄ H₇₄ O₁₁.

The empirical formula proposed for A-28086 factor D is C₄₄ H₇₄ O₁₁.Elemental analysis of factor D gave the following percentagecomposition: carbon, 67.59 percent; hydrogen 9.38 percent; oxygen, 22.77percent.

The theoretical percentage composition for C₄₄ H₇₄ O₁₁ is: carbon, 67.87percent; hydrogen, 9.51 percent; oxygen, 22.77 percent.

The infrared absorption spectrum of A-28086 factor D contains thefollowing observable absorption maxima: 2.89, 3.39, 3.43, 3.50, 5.88,6.90, 7.27, 7.60, 7.84, 9.00, 9.26, 9.62, 10.31, 10.58, 11.10, and 11.49microns.

A-28086 factor D in 95 percent aqueous ethanol shows no ultravioletabsorption.

The nuclear magnetic resonance spectrum of A-28086 factor D indeuterochloroform showed the following characteristics: δ 6.00, 4.20,4.10, 4.00, 3.98, 3.92, 3.86, 3.83, 3.79, 3.67, 3.64, 3.57, 3.54, 2.88,2.81, 2.71, 2.62, 2.58, 2.48, 2.43, 2.37, 2.29, 2.21, 2.15, 2.10, 2.04,1.97, 1.89, 1.83, 1.76, 1.68, 1.61, 1.58, 1.55, 1.47, 1.39, 1.30, 1.25,1.18, 0.95, 0.90, 0.88, 0.84, 0.74, and 0.68 ppm.

Antibiotic A-28086 factor D, crystallized from acetone-water, has thefollowing characteristic X-ray powder-diffraction pattern (Cu⁺⁺radiation, 1.5405λ, nickel filter, d = interplanar spacing inangstroms):

    ______________________________________                                                       Relative                                                       d              Intensity                                                      ______________________________________                                        12.40          100                                                            10.20          70                                                             8.85           90                                                             7.80           30                                                             6.80           10                                                             6.30           100                                                            5.70           20                                                             5.35           20                                                             5.10           20                                                             4.90           10                                                             4.65           20                                                             4.45           40                                                             4.20           30                                                             3.30           10                                                             3.15           10                                                             2.99           05                                                             2.77           05                                                             2.28           05                                                             ______________________________________                                    

The specific rotation of antibiotic A-28086 factor D is -56° (c = 0.1,methanol), when determined at a temperature of 25° C.

Electrometric titration of A-28086 factor D in 80 percent aqueousdimethylformamide indicated the presence of a titratable group with apK_(a) value of 8.67.

Antibiotic A-28086 factor D is soluble in a variety of organic solventssuch as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, ethylacetate, chloroform, acetone and benzene. A-28086 factor D is onlyslightly soluble in nonpolar organic solvents such as hexane and isinsoluble in water.

Antibiotic A-28086 factor D has an acid function capable of formingsalts and ester derivatives and at least one hydroxyl group capable ofesterification.

Based on the physical characteristics hereinabove recited, a structurefor antibiotic A-28086 factor D can be proposed. Since the structuredetermination is merely postulated, however, it is to be understood thatthe structure presented herein represents merely a working hypothesis.The tentative structure for A-28086 factor D is shown in Formula II:##STR2## wherein either: R₁ = CH₃ and R₂ = C₂ H₅ or: R₁ = C₂ H₅ and R₂ =CH₃

The R_(f) values of antibiotic A-28086 factors A, B and D in variouspaper-chromatographic systems, using Bacillus subtilis ATCC 6633 as adetection organism, are given in Table I.

                  TABLE I                                                         ______________________________________                                        R.sub.f Value                                                                 Factor A                                                                             Factor B Factor D Solvent System                                       ______________________________________                                        0.11   0.09     0.10     Water saturated with                                                          methyl isobutyl ketone                                                        (MIBK)                                               0.41   0.16     0.26     Water saturated with                                                          MIBK plus 2% p-toluene-                                                       sulfonic acid and 1%                                                          piperidine                                           0.54   0.46     0.36     Water:methanol:acetone                                                        (12:3:1)-adjusted to pH 10.5                                                  with NH.sub.4 OH and then low-                                                ered to pH 7.5 with H.sub.3 PO.sub.4                 0.48   0.36     0.29     1% MIBK, 0.5% NH.sub.4 OH in                                                  water                                                0.15   0.33     0.25     17.4 g K.sub.2 HPO.sub.4, 30 ml                                               ethanol per liter of                                                          water                                                0.24   0.51     0.26     Benzene saturated with                                                        water                                                0.24   0.11     0.09     Water                                                0.75   0.61     0.64     Water:MIBK:ethyl acetate                                                      (98:1:1)                                             ______________________________________                                    

In Table II are given the R_(f) values for antibiotic A-28086 factors A,B and D in two thin-layer-chromatographic systems on silica gel(precoated plates, E. Merck, Darmstadt, F-254, layer thickness 0.25 mm),again using B. subtilis ATCC 6633 as a detection organism.

                  TABLE II                                                        ______________________________________                                        R.sub.f Values                                                                Factor A                                                                             Factor B Factor D Solvent System                                       ______________________________________                                         0.24  0.42     0.26     Benzene-ethyl ace-                                                            tate (3:2)                                           0.54   0.34     0.66     Ethyl acetate-di-                                                             ethylamine (95:5)                                    ______________________________________                                    

Another substance, arbitrarily designated as A-28086-I, is co-producedwith the antibiotic A-28086 complex. Although A-28086-I is notmicrobiologically active, it is structurally related to the A-28086antibiotic factors. A-28086-I is a white crystalline compound (fromacetone-water) and has a melting point of about 160°-162° C. Comparativestudies of the NMR spectra and other properties of A-28086-I andsynthetically-prepared A-28086 factor A methyl ester give evidence thatA-28086-I is the methyl ester of A-28086 factor A or a closely relatedcompound such as a stereoisomer.

Although A-28086-I initially co-precipitates with the active A-28086antibiotic factors, it is readily separated from them by silica gelchromatography. A-28086-I has an approximate R_(f) value of 0.53 onsilica gel thin-layer chromatography with ethyl acetate as the elutingsolvent and using vanillin spray reagent (3% vanillin in methanol + 0.5ml conc H₂ SO₄ per 100 ml of solution) for detection. After sprayingwith vanillin and heating, A-28086-I gives a blue spot while the A-28086antibiotic factors give bright pink spots which quickly turn darkbrownish-blue.

Antibiotic A-28086 factors A and B and the specified acyl esterderivatives of factor A are capable of forming salts. Thephysiologically-acceptable alkali-metal, alkaline-earth-metal and aminesalts of antibiotic A-28086 factors A an B and the C₂ -C₆ -acyl esterderivatives of factor A are also part of this invention."Physiologically-acceptable" salts are salts which are alsopharmaceutically acceptable, that is, salts in which the toxicity of thecompound as a whole toward warm-blooded animals is not increasedrelative to the non-salt form. Representative and suitable alkali-metaland alkaline-earth-metal salts of A-28086 factors A and B include thesodium, potassium, lithium, cesium, rubidium, barium, calcium, andmagnesium salts. Suitable amine salts of A-28086 factors A and B includethe ammonium and the primary, secondary and tertiary C₁ -C₄-alkylammonium and hydroxy-C₂ -C₄ -alkylammonium salts. Illustrativeamine salts include those formed by reaction of A-28086 factors A and Bwith ammonium hydroxide, methylamine, sec-butylamine, isopropylamine,di-ethylamine, diisopropylamine, ethanolamine, triethylamine,3-amino-1-propanol and the like.

The alkali-metal and alkaline-earth-metal cationic salts of A-28086factors A and B and of the factor A acyl ester derivatives are preparedaccording to procedures commonly employed for the preparation ofcationic salts. For example, the free acid form of the antibiotic factoror ester derivative is dissolved in a suitable solvent such as warmmethanol or ethanol; a solution containing the stoichiometric quantityof the desired inorganic base in aqueous methanol is added to thissolution. The salt thus formed can be isolated by routine methods, suchas filtration or evaporation of the solvent.

The salts formed with organic amines can be prepared in a similarmanner. For example, the gaseous or liquid amine can be added to asolution of the antibiotic factor in a suitable solution such asacetone, and the solvent and excess amine can be removed by evaporation.

It is well known in the veterinary pharmaceutical art that the form ofan antibotic is not significant when treating an animal with theantibiotic. In most cases, conditions within the animal change the drugto forms other than the form in which it was administered. The salt formin which it may be administered is, therefore, insignificant to themethod of treatment. The salt form may, however, be chosen for reasonsof economics, convenience, and toxicity.

A-28086 factor A forms acyl ester derivatives. Esterification occurs atone of the hydroxyl groups of A-28086 factor A upon treatment with a C₂-C₆ -acid anhydride or acid chloride. Such esters are typically preparedby reacting A-28086 factor A with, for example, the corresponding acidanhydride at room temperature. These ester derivatives are also usefulas antibiotics and as agents which increase feed-utilization efficiency.

The following paragraphs describe the characteristics of theseA-28086-factor A acyl ester derivatives.

A-28086 factor A acetyl ester derivative is a white crystalline compound(from acetone-water) with a melting point of about 100°-103° C. A-28086factor A acetyl ester derivative has an empirical formula of C₄₅ H₇₄ O₁₂and a molecular weight of about 807, based on the empirical formulaproposed for A-28086 factor A. Elemental analysis of factor A acetylester derivative gave the following percentage composition:

Calcd. for C₄₅ H₇₄ O₁₂ : C, 66.97; H, 9.24; O, 23.79. Found: C, 67.67;H, 8.71; O, 23.13.

The infrared spectrum of A-28086 factor A acetyl ester derivative inchloroform is shown in FIG. 3 of the accompanying drawings. Thefollowing absorption maxima are observed: 2.85, 3.36, 3.38 (strong),5.80, 6.83, 7.25, 7.52 (strong), 7.60 (weak), 7.80 (strong), 8.45(strong), 8.80 (strong), 8.95 (strong), 9.10 (strong), 9.20, 9.63, 9.80(strong), 10.12 (weak), 10.25 (weak), and 10.50 microns.

The ultraviolet spectrum of A-28086 factor A acetyl ester in ethanolshows only end absorption.

Electrometric titration of A-28086 factor A acetyl ester derivative in80% aqueous dimethylformamide indicated the presence of a titratablegroup with a pK_(a) value of 8.5.

A-28086 factor A propionyl ester derivative is a white crystallinecompound (from acetone-water) with a melting point of about 96°-98° C.A-28086 factor A propionyl ester derivative has an empirical formula ofC₄₆ H₇₆ O₁₂ and a molecular weight of about 821, based on the empiricalformula proposed for A-28086 factor A. Elemental analysis of factor Apropionyl ester derivative gave the following percentage composition:

Calcd for C₄₆ H₇₆ O₁₂ : C, 67.29; H, 9.33; O, 23.38. Found: C, 66.06; H,9.17; O, 23.41.

The infrared spectrum of A-28086 factor A propionyl ester derivative inchloroform is shown in FIG. 4 of the accompanying drawings. Thefollowing absorption maxima are observed: 2.85, 3.33, 3.38 (strong),3.45 (strong), 5.75 (strong), 5.82, 6.81, 7.22, 7.30 (strong), 7.50(weak), 7.60 (weak), 7.80, 8.43, 8.75 (strong), 8.90, 9.05, 9.15(strong), 9.50 (strong), 9.63, 9.83 (weak), 10.05 (strong), 10.13, 10.20(strong), 10.45, and 10.68 microns.

The ultraviolet spectrum of A-28086 factor A propionyl ester derivativein ethanol shows only end absorption.

Antibiotic A-28086 factor A butyryl ester derivative is a whitecrystalline compound (from acetone-water) with a melting point of about96°-98° C. A-28086 factor A butyryl ester derivative has an empiricalformula of C₄₇ H₇₈ O₁₂, a molecular weight of about 835, and anapproximate composition of C, 67.60%, H; 9.41%; O, 22.99%, as derivedfrom the empirical formula proposed for A-28086 factor A.

The infrared spectrum of A-28086 factor A butyryl ester derivative inchloroform is shown in FIG. 5 of the accompanying drawings. Thefollowing absorption maxima are observed: 2.89, 3.40, 3.45, 3.51, 5.85,5.92 (strong), 5.97 (strong), 6.90, 7.30, 7.84 (weak), 8.55, 8.85(weak), 9.01 (strong), 9.26, 9.76, 9.95, 10.31, and 10.64 microns.

Antibiotic A-28086 factor A valeryl ester derivative is a whitecrystalline compound (from acetone-water) with a melting point of about173°-175° C. A-28086 factor A valeryl ester derivative has an empiricalformula of C₄₈ H₈₀ O₁₂, a molecular weight of about 849, and anapproximate composition of C, 67.89%; H, 9.50%; O, 22.61%, as derivedfrom the empirical formula proposed for A-28086 factor A.

The infrared spectrum of A-28086 factor A valeryl ester derivative inchloroform is shown in FIG. 6 of the accompanying drawings. Thefollowing absorption maxima are observed: 2.90, 3.40, 3.45, 3.51, 5.87,5.92 (strong), 5.99 (strong), 6.91, 7.30, 7.69 (weak), 7.87 (weak),8.16, 8.58, 8.85 (weak), 9.26, 9.76, 10.00 (weak), 10.31, and 10.64microns.

Antibiotic A-28086 factor A caproyl ester derivative is a whitecrystalline compound (from acetone-water) with a melting point of about163°-167° C. A-28086 factor A caproyl ester derivative has an empiricalformula of C₄₉ H₈₂ O₁₂, a molecular weight of about 863, and anapproximate composition of C, 68.18%; H, 9.58%; O, 22.24%, as derivedfrom the empirical formula proposed for A-28086 factor A.

The infrared spectrum of A-28086 factor A caproyl ester derivative inchloroform is shown in FIG. 7 of the accompanying drawings. Thefollowing absorption maxima are observed: 2.90, 3.40, 3.45, 3.51, 5.87,5.92 (strong), 5.97 (strong), 6.90, 7.30, 7.66 (weak), 7.84 (weak),8.16, 8.58, 8.85 (weak), 9.05 (strong), 9.17, 9.72, 9.95, 10.29, and10.62 microns.

Acylation of A-28086 factor A results in the following changes in the ¹H nuclear magnetic resonance spectrum: the carbinyl resonance occurringat about 4 ppm is shifted downfield to approximately 5.3 ppm (the exactposition varies slightly in the various acyl derivatives), and thevinyl-proton signals are also shifted. This is characteristic of thechange represented in partial structure by: ##STR3## wherein Rrepresents C₁ -C₅ -alkyl. This partial structure is in full accord with¹ H homonuclear decoupling experiments and ¹³ C nuclear magneticevidence from the acetyl and propionyl ester derivatives.

The C₂ -C₆ -acyl ester derivatives of A-28086 factor A are soluble in avariety of organic solvents such as methanol, ethanol,dimethylformamide, dimethyl sulfoxide, ethyl acetate, chloroform,acetone, and benzene; but are only slightly soluble in nonpolar organicsolvents such as hexane; and are insoluble in water.

Each of the C₂ -C₆ -acyl ester derivatives of A-28086 has an acidfunction capable of forming salts and ester derivatives.

The novel antibiotics of this invention are produced by culturing anA-28086-producing strain of Streptomyces aureofaciens under submergedaerobic conditions in a suitable culture medium until substantialantibiotic activity is produced. The antibiotics are recovered byemploying various isolation and purification procedures commonly usedand understood in the art.

One of the new organisms useful for the preparation of A-28086antibiotics was isolated from a soil sample collected on Mount Ararat inTurkey. This organism is classified as a strain of Streptomycesaureofaciens Duggar, as described by E. B. Shirling and D. Gottlieb in"Cooperative Description of Type Cultures of Streptomyces. III.Additional Species Descriptions from First and Second Studies," Intern.Bull. Systematic Bacteriol. 18, 279-392 (1968). This classification isbased on methods recommended for the International Streptomyces Project[E. B. Shirling and D. Gottlieb, "Methods for Characterization ofStreptomyces species," Intern. Bull. Systematic Bacteriol. 16, 313-340(1966)] along with certain supplementary tests. Color names wereassigned according to the ISCC-NBS method (K. L. Kelly and D. B. Judd,"The ISCC-NBS Method of Designating Color and a Dictionary of ColorNames," U.S. Dept. of Commerce, Circ. 553, 1955, Washington, D.C.).Figures in parentheses refer to the Tresner and Backus color series[Tresner, H. D. and S. J. Backus, "System of Color Wheels forStreptomyces Taxonomy," Appl. Microbiol. 11, 335-338 (1963)]; color tabdesignations are underlined. The Maerz and Paul color blocks (A. Maerzand M. R. Paul, "Dictionary of Color," McGraw-Hill Book Co., Inc., NewYork, N.Y., 1950) are enclosed in brackets. Cultures were grown at 30°C. for fourteen days unless otherwise noted.

CHARACTERIZATION OF A-28086-PRODUCING STRAIN NRRL 5758 Morphology

Sporulating aerial hyphae consist of hooks, loops and open spirals. Alsoobserved was morphology representative of the rectus flexibilis type.Spores are short and cylindrical and are in chains of 10-50 spores. Thespores measure 1.3μ × 1.75μ with a range of 1.3μ to 1.95 × 1.3μ. Thespore surface, as observed by electron microscopy, is smooth.

    ______________________________________                                        Cultural Characteristics of NRRL                                              5758 on Various Media                                                         Medium          Characteristics                                               ______________________________________                                        ISP #2 (yeast extract-                                                                        Growth-abundant; reverse mod-                                 malt extract)   erate yellow [11K3]; fair-to-                                                 good aerial mycelium and                                                      sporulation; white (W) 13 ba                                                  and dark gray (Gy) 3 ih;                                                      no soluble pigment.                                           ISP #3 (oatmeal)                                                                              Growth-good; reverse grayish                                                  yellow [11B2]; fair aerial                                                    mycelium (Gy) dark gray,                                                      3 ih; slight brown soluble                                                    pigment.                                                      ISP #4 (inorganic                                                                             Growth-abundant; reverse                                      salts-starch agar)                                                                            light yellow brown [12E5];                                                    aerial mycelium and spore                                                     (W) purplish white 13 ba                                                      to (GY) light gray d; no                                                      soluble pigment.                                              ISP #5 (glycerol-                                                                             Growth-good; reverse pale                                     asparagine agar)                                                                              yellow-green [10B1]; good                                                     aerial mycelium and spores                                                    (Gy) yellowish gray 2 dc,                                                     to light grayish reddish                                                      brown 5 fe; no soluble pig-                                                   ment.                                                         Tomato paste-oatmeal                                                                          Growth-abundant; reverse                                      agar            light yellow brown [13H7];                                                    fair to good aerial mycelium                                                  and spores (W) white a to                                                     (Gy) medium gray g; very                                                      slight brown soluble pigment.                                 Glycerol-glycine agar                                                                         Growth-abundant; reverse                                                      dark grayish yellow [12I6];                                                   good aerial mycelium and                                                      spores (Y) pale yellow                                                        2 db; no soluble pigment.                                     Glucose-asparagine agar                                                                       Growth-abundant; reverse                                                      grayish greenish yellow                                                       [12E2]; abundant aerial                                                       mycelium and spores (Gy)                                                      yellowish gray 2 dc; very                                                     slight brown soluble pig-                                                     ment.                                                         Nutrient agar   Growth-good; reverse gray-                                                    ish yellow [12B2]; aerial                                                     mycelium and spores, no                                                       color assignment because of                                                   poor growth; no soluble                                                       pigment.                                                      Bennett's agar  Growth-abundant, reverse                                                      grayish yellow [12K3];                                                        scant aerial mycelium and                                                     spores (Gy) yellowish gray                                                    2 dc; no soluble pigment.                                     Calcium malate agar                                                                           Growth-good; reverse gray-                                                    ish brown [15C8]; no aerial                                                   mycelium or sporulation;                                                      brown soluble pigment.                                                        Area by inoculum cleared.                                     Czapek's solution agar                                                                        Growth-poor; no color                                                         assignment due to poor                                                        growth.                                                       Emerson's agar  Growth-abundant; reverse                                                      grayish yellow [11J5]; no                                                     aerial mycelium or spores;                                                    no soluble pigment.                                           Tyrosine agar   Growth-abundant; reverse                                                      light olive brown [14C4];                                                     abundant aerial mycelium                                                      and spores from (W) b                                         (center) to (Gy) light                                                                        brownish gray 3 fe (margin);                                                  very slight brown soluble                                                     pigment.                                                      Tryptone-yeast agar                                                                           Growth-scant; no color                                                        assignment.                                                   ______________________________________                                    

The NRRL 5758 organism was studied for selected physiological propertiesin accordance with standard procedures. The properties observed andcharacteristics found were as follows:

    ______________________________________                                        Property Observed                                                                              Characteristic                                               ______________________________________                                        Action on milk   Milk peptonized, white                                                        growth ring; cleared area                                                     tannish yellow-pH reaction                                                    5.7                                                          Nitrate reduction                                                                              Positive                                                     Nutrient gelatin 30% liquefaction at 14                                                        days                                                         Melanin pigment                                                               production on:                                                                Tyrosine-agar    Very weak positive (pig-                                     slants           ment after 4 days)                                           Difco peptone    Negative                                                     yeast extract                                                                 Iron agar slants                                                              Tryptone-yeast-  Negative                                                     extract broth                                                                 Temperature require-                                                                           26-30° C-good growth; 30-                             ments (ISP medium #2                                                                           37° C.-excellent growth                               yeast extract malt                                                                             and sporulation; 45° C.-                              extract slants)  slight vegetative growth;                                                     reddish soluble pigment.                                     ______________________________________                                    

The results of carbon utilization tests carried out with organism NRRL5758 are set forth below. The symbols used to indicate growth responseare:

+ good growth, positive utilization

(+) poor to fair growth

(-) faint growth, probably no utilization

- no growth, no utilization

    ______________________________________                                        Carbon Source         Response                                                ______________________________________                                        D-glucose             +                                                       L-arabinose           +                                                       D-xylose              +                                                       D-fructose            +                                                       sucrose               -                                                       D-mannitol            -                                                       i-inositol            +                                                       rhamnose              +                                                       raffinose             -                                                       -C control                                                                     (no carbohydrate)    -                                                       ______________________________________                                    

A second new A-28086-producing organism was derived from S. aureofaciensNRRL 5758 by a series of natural selections, followed by chemicalmutation. This organism, identified as NRRL 8092, is also classified asa strain of Streptomyces aureofaciens Duggar. This classification wasbased on studies of organism NRRL 8092 using the earlier-describedStreptomyces classification methods and similar growth conditions. Thecharacteristics of organism NRRL 8092 observed in these studies aresummarized in the following paragraphs.

CHARACTERIZATION OF A-28086-PRODUCING STRAIN NRRL 8092 Morphology

On medium ISP #7 (tyrosine agar) the culture produces occasional hooks,but mainly produces short, straight sporophores. Spore chains are lessthan 10 spores per chain, usually 4-7 spores per chain. Short straightspore chains were observed in the following media: ISP #3,Czapek's-solution agar and ISP #5. Abundant coremia were observed onEmerson's agar. Electron microscope observations were made on tyrosineagar (ISP #7) and glucose-asparagine agar. Spores are smooth and rangein size from 1.2 to 2.0μ in length and about 1.0μ in diameter. Theaverage spore size is 1.6μ × 1.0μ.

    ______________________________________                                        Cultural Characteristics of NRRL                                              8092 on Various Media                                                         Medium          Characteristics                                               ______________________________________                                        ISP #2 (yeast extract-                                                                        Growth-fair; reverse light                                    malt extract)   yellow brown [12H8]; fair                                                     aerial mycelium; poor                                                         sporulation; aerial pale                                                      gray [11A1]; no soluble                                                       pigment.                                                      ISP #3 (oatmeal)                                                                              Growth-sparse; reverse hya-                                                   line; no aerial mycelium;                                                     no soluble pigment.                                           ISP #4 (inorganic                                                                             Growth-moderate; reverse                                      salts-starch agar)                                                                            grayish yellow [11B2]; scant                                                  aerial mycelium and sporula-                                                  tion; aerial pale yellow                                                      gray [10A1]; no soluble pig-                                                  ment.                                                         ISP #5 (glycerol-                                                                             Growth-moderate; reverse                                      asparagine agar)                                                                              pale yellow [10F2]; fair                                                      aerial mycelium; scant sporu-                                                 lation; aerial white [10A1];                                                  no soluble pigment.                                           Tomato paste-oatmeal                                                                          Growth-moderate; reverse gray-                                agar            ish green-yellow; aerial                                                      mycelium fair; moderate                                                       sporulation, light pale gray                                                  [53A2]; no soluble pigment.                                   Glycerol-glycine agar                                                                         Growth-abundant; reverse                                                      grayish yellow [11E4]; mod-                                                   erate aerial mycelium, white                                                  [10A1]; no sporulation; no                                                    soluble pigment.                                              Glucose-asparagine agar                                                                       Growth-moderate; reverse pale                                                 yellow [10F2]; moderate                                                       aerial mycelium and sporula-                                                  tion, white [10A1]; no soluble                                                pigment.                                                      Nutrient agar   Growth-sparse; reverse pale                                                   yellow [10B2]; no aerial                                                      mycelium; no soluble pig-                                                     ment.                                                         Bennett's agar  Growth-fair; reverse medium                                                   yellow pink [11A7]; very                                                      scant aerial mycelium; no                                                     sporulation; no soluble pig-                                                  ment.                                                         Calcium malate agar                                                                           Growth-very scant, hyaline;                                                   no aerial mycelium; no solu-                                                  ble pigment.                                                  Czapek's solution agar                                                                        Growth-very scant; reverse                                                    hydaline; no aerial mycelium;                                                 no soluble pigment.                                           Emerson's agar  Growth-moderate; reverse                                                      grayish yellow [11I5]; spotty                                                 aerial mycelium; no sporula-                                                  tion; no soluble pigment.                                     Tyrosine agar   Growth-moderate; reverse light                                                yellow brown [12H6]; moderate                                                 aerial mycelium, light pale                                                   gray margin [53A2], center                                                    near white, and moderate                                                      sporulation; no soluble pig-                                                  ment.                                                         Tryptone-yeast agar                                                                           Growth-very scant, hyaline; no                                                aerial mycelium; no soluble                                                   pigment.                                                      ______________________________________                                    

Organism NRRL 8092 was also studied for selected physiologicalproperties in accordance with standard procedures. The propertiesobserved and characteristics found were as follows:

    ______________________________________                                        Property Observed                                                                              Characteristic                                               ______________________________________                                        Action on milk   peptonized (90%); pale-                                                       yellow growth ring, cleared                                                   area pale yellow--pH reaction                                                 4.6                                                          Nitrate reduction                                                                              Positive                                                     Nutrient gelatin 50% hydrolyzed at 14 days                                    Melanin pigment                                                               production on:                                                                 Tyrosine-agar   Very weakly positive                                          slants                                                                        Tryptone-yeast- Negative                                                      extract broth                                                                 Carrot plug     Abundant growth, pale                                                         yellow; no aerial mycelium                                    Potato plug     Abundant growth, grayish                                                      white; no aerial mycelium;                                                    no change in plug.                                           Temperature require-                                                                           25° C.-Abundant growth; fair                          ments (ISP medium #2                                                                           aerial mycelium; reverse                                     yeast extract malt                                                                             light brown; no soluble pig-                                 extract slants)  ment.                                                                         30° C.-Abundant growth; fair                                           aerial mycelium; reverse                                                      light brown; no soluble pig-                                                  ment.                                                                         37° C.-Abundant growth; fair                                           aerial mycelium; reverse-brown;                                               soluble pigment brown.                                                        40° C.-Abundant growth; sparse                                         aerial mycelium; reverse red                                                  brown; soluble pigment deep                                                   red brown.                                                                    45° C.-Fair growth; no aerial                                          mycelium; reverse red brown;                                                  moderate red brown pigment.                                  ______________________________________                                    

The results of carbon utilization tests carried out with organism NRRL8092 are set forth below. The symbols used to indicate growth responseare:

+ good growth, positive utilization

(+) poor to fair growth

(-) faint growth, probably no utilization

- no growth, no utilization

    ______________________________________                                        Carbon Source         Response                                                ______________________________________                                        D-glucose             +                                                       L-arabinose           +                                                       D-xylose              +                                                       D-fructose            +                                                       sucrose               -                                                       D-mannitol            -                                                       i-inositol            +                                                       rhamnose              +                                                       raffinose             -                                                       -C control                                                                     (no carbohydrate)    -                                                       ______________________________________                                    

Certain characteristics of the A-28086-producing S. aureofaciens strainsdiffer from the characteristics of the organism described by Shirlingand Gottlieb. These differences are summarized in Table III:

                  TABLE III                                                       ______________________________________                                        Carbon                          Published                                     utilization                                                                             NRRL 5758  NRRL 8092  Description                                   ______________________________________                                        sucrose   -          -          +                                             i-inositol                                                                              +          +          -                                             rhamnose  +          +          -                                             Gelatin   30% in 14  50% in 14  Limited or                                    Liquifaction                                                                            days       days       None                                          Action on Milk                                                                          Milk pep-  Milk pep-  Limited and                                             tonized,   tonized,   variable pep-                                           white      pale-yel-  tonization                                              growth     low        (often none);                                           ring       growth     limited growth                                                     ring       and coagulation                               ______________________________________                                    

The characteristics of organism NRRL 5758 which differ from thecharacteristics of organism NRRL 8092 are summarized in Table IV.

                  Table IV                                                        ______________________________________                                        Characteristic                                                                             NRRL 5758    NRRL 8092                                           ______________________________________                                        Vegetative Color                                                                           Yellow on    Cream to pale-                                                   several      yellow on several                                                media        media                                               Sporulation  Some spiral  Short straight                                                   sporophores  sporophores with                                                 on tomato-   occasional hooks                                                 paste:oatmeal                                                                 and inorganic                                                                 salts-starch                                                                  media                                                            Growth on:                                                                    Calcium malate                                                                             Growth fair, Growth sparse,                                                   brown; with  clear, no clearing                                               clearing                                                         Inorganic salts-                                                                           Moderate spor-                                                                             Scant sporulation;                                  starch       ulation;     aerial pale yellow                                               aerial pur-  gray                                                             plish white                                                                   to gray.                                                         Bennett's agar                                                                             Reverse pale-                                                                              Reverse pink                                                     yellow                                                           ______________________________________                                    

The Streptomyces aureofaciens cultures useful for production of A-28086antibiotics have been deposited and made a part of the stock culturecollection of the Northern Marketing and Nutrition Research Division,U.S. Dept. of Agriculture, Agricultural Research Service, Peoria,Illinois, 61604, from which they are available to the public under thenumbers NRRL 5758 and NRRL 8092.

The culture medium employed to grow Streptomyces aureofaciens NRRL 5758or Streptomyces aureofaciens NRRL 8092 can be any one of a number ofmedia. For economy in production, optimal yield, and ease of productisolation, however, certain culture media are preferred. Thus, forexample, preferred carbohydrate sources in large-scale fermentation aretapioca dextrin and sucrose, although glucose, corn starch, fructose,mannose, maltose, lactose, and the like can also be employed. Corn oil,peanut oil, soybean oil and fish oil are other useful sources of carbon.A preferred nitrogen source is enzyme-hydrolyzed casein, althoughpeptones, soybean meal, cotton-seed meal, amino acids such as glutamicacid, and the like are also useful. Among the nutrient inorganic saltswhich can be incorporated in the culture media are the customary solublesalts capable of yielding sodium, magnesium, calcium, ammonium,chloride, carbonate, sulfate, nitrate, and like ions.

Essential trace elements necessary for the growth and development of theorganism should also be included in the culture medium. Such traceelements commonly occur as impurities in other constituents of themedium in amounts sufficient to meet the growth requirements of theorganism.

It may be necessary to add small amounts (i.e. 0.2 ml/l.) of an antifoamagent such as polypropylene glycol to large-scale fermentation media iffoaming becomes a problem.

Although it is not essential, the antibiotic production of theA-28086-producing Streptomyces aureofaciens strains is enhanced by theaddition of a small amount of oil such as soybean oil.

For production of substantial quantities of the A-28086 antibiotics,submerged aerobic fermentation in tanks is preferred. Small quantitiesof the A-28086 antibiotics may be obtained by shake-flask culture.Because of the time lag in antibiotic production commonly associatedwith innoculation of large tanks with the spore form of the organism, itis preferable to use a vegetative inoculum. The vegetative inoculum isprepared by inoculating a small volume of culture medium with the sporeform or mycelial fragments of the organism to obtain a fresh, activelygrowing culture of the organism. The vegetative inoculum is thentransferred to a larger tank. The medium used for the growth of thevegetative inoculum can be the same as that employed for largerfermentations, but other media can also be employed.

The A-28086-producing organisms can be grown at temperatures betweenabout 20° and about 40° C. Optimum A-28086 production appears to occurat temperatures of about 27°-30° C.

As is customary in aerobic submerged culture processes, sterile air isblown through the culture medium. For efficient growth of the organismthe volume of air employed in the tank production is preferably above0.1 volume of air per volume of culture medium per minute. For efficientproduction of the A-28086 antibiotics the volume of air employed in thetank production is preferably above 0.25 volume of air per volume ofculture medium per minute. High levels of dissolved oxygen do notdepress antibiotic production.

The production of antibiotics can be followed during the fermentation bytesting samples of the broth or of extracts of the mycelial solids forantibiotic activity against organisms known to be sensitive to theantibiotics. One assay organism useful in testing the antibiotics of thepresent invention is Bacillus subtilis ATTCC 6633. The bioassay isconveniently performed by paper-disc assay on agar plates.

The initial pH of the uninoculated culture medium varies with the mediumused. In general, the pH should be in the range of 6.0 to 7.5. Theharvest pH at the end of the fermentation is usually slightly higher, inthe range of 6.5 to 8.0.

Generally, antibiotic activity is detectable on the second day of thefermentation. Maximum production of antibiotic activity usually occursbetween about the sixth and the tenth days.

Following their production under submerged aerobic fermentationconditions, the A-28086 antibiotics previously described can berecovered from the fermentation medium by methods commonly employed inthe fermentation art. The antibiotic activity produced duringfermentation of an A-28086-producing organism occurs in both themycelial mass and in the filtered broth. Maximum recovery of the A-28086antibiotics is accomplished, therefore, by a combination of methods,including filtration, extraction, and adsorption chromatography. Apreferred solvent for separating the A-28086 antibiotics from eitherwhole or filtered fermentation broth is ethyl acetate, although othercommonly used solvents are satisfactory.

An especially advantageous method of separating the A-28086 factors A, Band D is to lower the pH of the whole fermentation broth to about pH3.0. At this pH 3.0 the A-28086 factors A, B, and D are convenientlyseparated with the mycelial mass by filtration. This method is thesubject of a copending application of Boeck and Berg titled ANTIBIOTICRECOVERY PROCESS, Ser. No. 569,712, filed this even date herewith.Another advantageous method of separating the A-28086 factors involvesadding a bicarbonate such as, for example, sodium bicarbonate, to thewhole broth in amounts of approximately one gram per liter. The A-28086factors are, thereby, conveniently separated with the mycelial mass insalt form. Methanol is a preferred solvent for separating theantibiotics from the mycelial mass, but other lower alcohols and ketonesare also suitable.

Azeotropic distillation can also be advantageously employed in therecovery of the A-28086 antibiotics. In this method an organic solventwhich forms an appropriate azeotrope with water is added to the aqueousfermentation broth. This solvent-broth mixture is subjected toazeotropic distillation in order to remove at least half the water fromthe broth, leaving a water-solvent mixture in which the A-28086antibiotics are in solution in the organic solvent. Insolubleby-products can be separated by suitable means such as filtration orcentrifugation. The A-28086 antibiotics can then be recovered from theorganic solution by well-known procedures such as evaporation ofsolvent, precipitation by adding a nonsolvent, or extraction.

Organic solvents which form appropriate azeotropes with water in orderto carry out such a recovery procedure include, illustratively, butylalcohol, amyl alcohol, hexyl alcohol, benzyl alcohol, butyl acetate,amyl acetate, 1,2-dichloroethane, 3-pentanone, 2-hexanone, benzene,cyclohexanone, toluene, the xylenes and the like.

There is special advantage in recovery by azeotropic distillation onlarge-scale fermentation processes. Both water and solvent takenoverhead in the azeotrope can be separated by known techniques andthereafter recycled for further use. The water thus removed is free ofcontaminants and does not require a waste disposal process. The solventthus removed may be recycled to the process.

Further purification of the A-28086 antibiotics includes additionalextraction and adsorption procedures. Adsorptive materials such assilica gel, carbon, Florisil® (magnesium silicate, Floridin Co., P.O.Box 989, Tallahassee, Fla.) and the like can be advantageously employed.

Alternatively, the culture solids, including medium constituents andmycelium can be used without extraction or separation, but preferablyafter removal of water, as a source of the A-28086 antibiotics. Forexample, after production of A-28086 antibiotic activity, the culturemedium can be dried by lyophilization and mixed directly into feedpremix.

In another aspect, after production of A-28086 activity in the culturemedium, the mycelium can be separated and dried to give a product whichcan be used directly in a feed premix. When separating the mycelium forsuch use, the addition of calcium carbonate (about 10 g./l.) aids infiltration and gives an improved dried product.

Under the conditions employed thus far, the Streptomyces aureofaciensstrains described previously and designated as NRRL 5758 and NRRL 8092produce antibiotic A-28086 factor A as the predominant factor. Althoughthe ratio of factors varies depending on the fermentation conditionsused, in general factor A accounts for more than 99 percent of the totalrecovered antibiotic activity from NRRL 5758 and for about 90 percent ofthe total recovered antibiotic activity from NRRL 8092. A-28086 factor Baccounts for most of the remaining antibiotic activity from NRRL 5758,and factor D is a minor factor. On the other hand, A-28086 factor Daccounts for about 8-10 percent of the total recovered antibioticactivity from NRRL 8092, and factor B is a minor factor.

Antibiotic A-28086 factors A, B, and D are separated from each other andare isolated as individual compounds by the use of well-known methodssuch as column chromatography, thin-layer chromatography and the like.For example, column chromatography over silica gel is used to separatefactors A, B, and D by eluting the column with varying solvent mixtures,such as benzene-ethyl acetate. Using benzene-ethyl acetate solventmixtures over a silica gel column, factor B is eluted first, and factorsA and D are eluted later. Thin-layer chromatography, as describedhereinabove, is a convenient method for monitoring elution progress.

The A-28086 compounds of this invention inhibit the growth of bacteriaand fungi which are pathogenic to animal and plant life. The relativemicrobiological activities of A-28086 factors A and B are describedbelow in Table V.

The test method was the conventional disc-diffusion method (6 mm padswere dipped in solutions containing 1 mg of compound/ml of solution;pads were placed on agar plates seeded with test organism).

                  TABLE V                                                         ______________________________________                                                        Zone of                                                                       Inhibition (mm)                                                                 A28086      A28086                                          Test Organism     factor A    factor B                                        ______________________________________                                        Staphylococcus aureus                                                                           19          21                                              Bacillus subtilis                                                             ATCC6633          28          31                                              Sarcina lutea ATCC9341                                                                          26          16                                              Mycobacterium avium                                                           ATCC7992          12          12                                              Saccharomyces pastorianum                                                     ATCC2366          17          --                                              Candida tropicalis                                                                              14          14                                              Fusarium moniliforme                                                                            12          Not Tested                                      ______________________________________                                    

In one important aspect, the A-28086 compounds inhibit the growth ofanaerobic bacteria. The minimal inhibitory concentrations (MIC) at whichA-28086 factor A inhibits various anaerobic bacteria, determined bystandard agar-dilution assay, are summarized in Table VI. Endpoints wereread after 24-hour incubation.

                  TABLE VI                                                        ______________________________________                                        Test Organism          MIC (μg/ml)                                         ______________________________________                                        Actinomyces bovis      <0.5                                                   Clostridium inocuum    <0.5                                                   Clostridium perfringens                                                                              <0.5                                                   Clostridium ramosum     4.0                                                   Clostridium septicum   <0.5                                                   Clostridium septicum bovine                                                                          <0.5                                                   Eubacterium aerofaciens                                                                               1.0                                                   Peptococcus anaerobius <0.5                                                   Peptostreptococcus intermedius                                                                        16.0                                                  Propionibacterium acnes 44                                                                            16.0                                                  Propionibacterium acnes 79                                                                            2.0                                                   Bacteroides fragilis ssp. fragilis                                                                    8.0                                                   Bacteroides fragilis ssp.                                                     thetaiotacmicron        8.0                                                   Bacteroides fragilis ssp. vulgatis                                            #1563                   8.0                                                   Bacteroides fragilis ssp.                                                     vulgatis 1211           2.0                                                   Fusobacterium symbiosum                                                                              <0.5                                                   Fusobacterium necrophorum                                                                             8.0                                                   Veillonella alcalescens                                                                               16.0                                                  ______________________________________                                    

The C₂ -C₆ -acyl ester derivatives of A-28086 factor A also haveantibacterial and antifungal activity. In one aspect, these derivativeshave increased gram-positive activity. The relative gram-positiveactivities of certain of these derivatives were compared with theactivity of factor A. The compounds were tested by turbidometric assayon a semiautomated system (Autoturb® microbiological assay system,Elanco) described by N. R. Kuzel and F. W. Kavanaugh in J. Pharmaceut.Sci. 60 (5), 764 and 767 (1971). In testing the A-28086 antibiotics, thefollowing test parameters were used: Staphylococcus aureus (H-Heatley)NRRL B-314 in a nutrient broth medium (pH 7), incubated for four hoursat 37° C. Test samples and the standard were dissolved in methanol-water(10:90). The standard, A-28086 factor A, was presented to the Autoturb®carrousel at concentrations of 2, 3, 4, and 5 mcg/ml. Test compoundswere diluted to contain approximately 3 or 4 mcg of activity per ml, aspresented to the carrousel. The relative activities of test compounds,as compared to that of the standard, are given below:

    ______________________________________                                                             Relative G+                                              Compound             Activity                                                 ______________________________________                                        A28086 factor A                                                               (standard)           1                                                        A28086-A acetyl ester                                                         derivative           2.5                                                      A28086-A propionyl                                                            ester derivative     7                                                        A28086-A n-butyryl                                                            ester derivative     8                                                        A28086-A n-valeryl                                                            ester derivative     14                                                       A28086-A n-caproyl                                                            ester derivative     20                                                       ______________________________________                                    

Activity against Mycoplasma is another useful aspect of theantimicrobial activity of the A-28086 compounds. Mycoplasma species,also known as pleuropneumonia-like (PPLO) organisms, are pathogenic toman and various animals. Agents active against PPLO organisms areespecially needed by the poultry industry. The minimal inhibitoryconcentrations (MIC) of antibiotic A-28086 factor A against variousMycoplasma species, as determined by in vitro broth-dilution studies,are summarized in Table VII below:

                  TABLE VII                                                       ______________________________________                                        Organism             MIC (mcg/ml)                                             ______________________________________                                        M. gallisepticum     12.5                                                     M. hyorhinis         12.5                                                     M. synoviae          6.25                                                     M. hypopneumoniae    6.25                                                     M. hyosynoviae       6.25                                                     ______________________________________                                    

In one embodiment, therefore, this invention provides an anti-PPLOagent. Solutions containing as little as 10 micrograms of antibioticA-28086 per milliliter of solution are useful for disinfecting surfacesto protect animals from various Mycoplasma species.

The A-28086 compounds also are antiviral agents. A-28086 factor A isactive against type III poliovirus, vaccinia virus, herpes virus andSemliki Forest virus, as demonstrated by in vitro plaque suppressiontests, similar to that described by Siminoff, Applied Microbiology 9[1], 66-72 (1961). A-28086 factor A is also active against TransmissibleGastro-enteritis virus, Newcastle Disease virus, and Infectious BovineRhinotracheitis virus, as demonstrated by similar tissue-culture tests.

In one aspect of this invention, therefore, an A-28086 compound can beadministered orally, topically or parenterally to mammals for thecontrol of viruses. Useful dosage levels for prevention or treatment ofviral disease vary from about 1 to about 5 mg/kg of mammalian bodyweight, depending upon the virus and upon whether the drug is to be usedprophylactically or therapeutically.

Furthermore, solutions containing an A-28086 compound, preferablytogether with a surfactant, can be used to decontaminate the in vitrohabitat on which viruses, such as polio or herpes, are present.Solutions containing from about 1 to about 1500 mcg/ml of an A-28086compound are effective in the control of viruses.

The acute toxicity of antibiotic A-28086 factor A, administeredintraperitoneally to mice and expressed as LD₅₀, is 7.15 mg/kg.

The A-28086 compounds of this invention are also insecticides andacaricides. The A-28086 compounds are active against insects such asMexican bean beetle, milkweed bug, and house fly and against mites suchas two-spotted spider mite when applied at rates as low as 500 ppm. Inaddition, the A-28086 compounds are active against mosquito larvae whenapplied at rates as low as one ppm.

Anticoccidial activity is an important property of the A-28086 compoundsof this invention. For example, feeding experiments show that an A-28086compound, when present in the feed of young chickens at levels as low as0.003 percent, improves weight gains and, at levels as low as 0.005percent, prevents mortality and decreases the number of lesions inchicks which have been challenged with coccidia. The results of testswith factor A in chicks challenged with various Eimeria species areshown in Tables VIII through X.

                                      TABLE VIII                                  __________________________________________________________________________    ACTIVITY OF ANTIBIOTIC A-28086 FACTOR A AGAINST EIMERIA TENELLA.              Drug Level in                       Percent                                   Feed, Percent                                                                          No. Of                                                                             Percent                                                                             Percent Average Reduction in                              by Weight                                                                              Chickens                                                                           Mortality                                                                           Weight Gain                                                                           Lesion Score                                                                          Lesion Score                              __________________________________________________________________________    0.04     10   0     50      0       100                                       0.02     10   0     82      0.2     95                                        0.01     10   0     94      0.2     95                                        Infected                                                                      Controls 20   35    68      3.65    --                                        Normal                                                                        Controls 20   0     100     --      --                                        0.02     20   0     84      0       100                                       0.01     20   0     96      0       100                                       0.005    20   0     91      2.8     27                                        0.0025   20   10    87      3.85    0                                         Infected                                                                      Controls 20   40    74      3.85    --                                        Normal                                                                        Controls 20   0     100     --      --                                        __________________________________________________________________________

                                      TABLE IX                                    __________________________________________________________________________    ACTIVITY OF ANTIBIOTIC A-28086 FACTOR A AGAINST VARIOUS EIMERIA               SPECIES.sup.1                                                                                                             Oocysts Passed                               Drug Level in  Percent                                                                            Average                                                                            Percent Total/                                       Feed, Percent                                                                          Percent                                                                             Weight                                                                             Lesion                                                                             Reduction in                                                                          Bird   Percent                    Infecting Organism                                                                       by Weight                                                                              Mortality                                                                           Gain Score                                                                              Lesion Score                                                                          (1 × 10.sup.6)                                                                 Reduction                  __________________________________________________________________________    Eimeria acervulina                                                                       0.005    0     87   0.75 53      6.15   83                                     0.0025  0     82   1.25 --      4.91   86                         Infected Controls                                                                        --       0     63   1.60 --      36.32  --                         Eimeria maxima                                                                           0.005    0     85   0.2  85      1.95   40                         Infected Controls                                                                        --       0     65   1.3  --      3.24   --                         Emeria brunetti                                                                          0.005    0     88   1.2  --      2.31   58                         Infected Controls                                                                        --       0     62   1.7  --      5.55   --                         __________________________________________________________________________     .sup.1 Four replicates, 5 broiler cockerels each                         

                                      TABLE X                                     __________________________________________________________________________    ACTIVITY OF ANTIBIOTIC A-28086 FACTOR A AGAINST MIXED EIMERIA                 SPECIES.sup.1                                                                            Drug Level   Percent                                                                            Intestinal Lesions                                                                       Cecal Lesions                                    In Percent                                                                           Percent                                                                             Weight    Percent    Percent                          Infecting Organism                                                                       by Weight                                                                            Mortality                                                                           Gain Average                                                                            Reduction                                                                           Average                                                                            Reduction                        __________________________________________________________________________    E. necatrix and                                                               E. tenella 0.005  0     94   0.7  59    1.75 50                               Infected Controls 20    52   1.7  --    3.5  --                               E. tenella,                                                                   E. necatrix,                                                                  E. maxima,                                                                    E. brunetti,                                                                  E. acervulina,                                                                and E. mivati                                                                            0.01   0     91   0.4  79    0.9  73                               Infected Controls                                                                        --     50    12   1.9  --    3.3  --                               __________________________________________________________________________     .sup.1 Four replicates, 5 broiler cockerels each.                        

For the prevention or treatment of coccidiosis in poultry, a non-toxicanticoccidial amount of an A-28086 compound is administered to birds,preferably orally on a daily basis. The A-28086 compound can be suppliedin many ways, but it is most conveniently supplied with aphysiologically-acceptable carrier, preferably the feed ingested by thebirds. Although a variety of factors must be considered in determiningan appropriate concentration of A-28086 compound, the rates ofadministration will be generally in the range of 0.003 to 0.04 percentby weight of unmedicated feed, and preferably in the range of 0.005 to0.02 percent.

The ability to improve feed-utilization efficiency in animals is anotherimportant property of A-28086 compounds. For example, A-28086 compoundsimprove feed-utilization efficiency in ruminants which have a developedrumen function.

As discussed hereinabove, efficiency of carbohydrate utilization inruminants is increased by treatments which stimulate the amimal's rumenflora to produce propionate compounds rather than acetate or butyratecompounds. The efficiency of feed use can be monitored by observing theproduction and concentration of propionate compounds in the rumen, usingthe following methods:

Remen fluid is obtained from a steer with a surgically-installed fistulaopening into the rumen. The steer is maintained on a high-grain ration.A sample of rumen fluid is strained through four layers of cheesecloth,and the filtrate is collected. The particulate matter retained by thecheesecloth is resuspended in enough physiological buffer to return itto the original volume of the rumen fluid, and this suspension isstrained again. The buffer used has the following composition:

    ______________________________________                                               Ingredient      g/liter                                                ______________________________________                                               Na.sub.2 HPO.sub.4                                                                            0.316                                                         KH.sub.2 PO.sub.4                                                                             0.152                                                         NaHCO.sub.3     2.260                                                         KCl             0.375                                                         NaCl            0.375                                                         MgSO.sub.4      0.112                                                         CaCl.sub.2 . 2H.sub.2 O                                                                       0.050                                                         FeSO.sub.4 . 7H.sub.2 O                                                                       0.008                                                         MnSO.sub.4 . H.sub.2 O                                                                        0.004                                                         ZnSO.sub.4 . 7H.sub.2 O                                                                       0.004                                                         CuSO.sub.4 . 5H.sub.2 O                                                                       0.002                                                         CoCl.sub.2 . 6H.sub.2 O                                                                       0.001                                                  ______________________________________                                    

as described by Cheng et al. in J. Dairy Sci. 38, 1225-1230 (1955).

The two filtrates are combined and allowed to stand until particulatematter separates to the top. The clear layer is separated, diluted withthe same buffer (1:1) and then adjusted to between pH 6.8 to 7.0.

The diluted rumen fluid (10 ml) is placed in a 25-ml flask with 40 mg ofthe above-described feed, an additional 1 mg of soybean protein, and thecompound to be tested. Four replicate flasks are used per treatment. Twosets of four control flasks each are also employed. A zero-time controland an incubated 16-hour control are used. All test flasks are incubatedfor 16 hours at 38° C. After incubation the pH is measured, and 25percent metaphosphoric acid (2 ml) is added to each flask. The samplesare allowed to settle, and the supernatants are analyzed by gaschromatography for propionate, acetate, and butyrate compounds. Activecompounds significantly increase propionate production over that ofcontrols.

Test-compound results are statistically compared with control results.Table XI shows the ratio of volatile-fatty-acid concentrations intreated flasks to concentrations in control flasks.

                  TABLE XI                                                        ______________________________________                                                       Ratio of Treated to Control                                                                 Molar Molar                                                  ppm in   Molar   %     %     Total                                            rumen    %       buty- pro-  VFA                                  Compound    fluid    acetate rate  pionate                                                                             mM/l                                 ______________________________________                                        A-28086 Factor A                                                                          1        0.94    0.87  1.34  1.26                                 A-28086 Factor B                                                                          1        0.97    0.96  1.15  1.29                                 A-28086 Factor A-                                                             Acetyl Ester                                                                              1        0.79    0.76  2.04  1.04                                 A-28086 Factor A-                                                             Propionyl Ester                                                                           1        0.90    0.68  1.73  1.25                                 A-28086 Factor A                                                              Butyryl Ester                                                                             1        0.91    0.81  1.56  1.19                                 A-28086 Factor A-                                                             Valeryl Ester                                                                             1        0.92    0.79  1.55  1.18                                 A-28086 Factor A-                                                             Caproyl Ester                                                                             1        0.86    0.97  1.57  0.99                                 ______________________________________                                    

Efficiency of carbohydrate utilization is further demonstrated by invivo tests performed in animals which have had fistulas installed intheir rumens, making it possible to withdraw specimens of the contentsof the rumen.

The test reported in Table XII was conducted with mature fistulatedsteers weighing about 1,000 lbs. each. Two steers were fed a normaldiet, and four steers in each treatment group were fed an identical dietto which A-28086 factor A had been added. To each aliquot (100 ml) ofrumen fluid taken, 10 percent metaphosphoric acid (100 ml) was added.The samples were allowed to settle, and the supernatants were analyzedby gas chromatographic methods to determine propionic acidconcentrations.

The results in Table XII are the mean percent increases in ruminalpropionic acid concentration, averaged over five analyses in a 14-daytreatment period. Controls had approximately 20 molar percent propionicacid.

                  TABLE XII                                                       ______________________________________                                        A-28086 factor A                                                                              Percent Increase                                              (mg/day)        Over Control                                                  ______________________________________                                         25             17.4                                                          100             54.0                                                          ______________________________________                                    

Furthermore, in vivo testing demonstrated that A-28086 factor Aincreases feed-utilization efficiency in sheep. The results of thesetests, conducted over a period of 56 days, are summarized in Table XIII.

                  TABLE XIII                                                      ______________________________________                                        A-28086-                    Feed                                              Factor A (g)                                                                           Number   Average   Intake      Percent                               per ton  of       Daily Gain                                                                              (lbs./                                                                              Feed/ Improve-                              of feed  Animals  (lbs./day)                                                                              day)  Gain  ment                                  ______________________________________                                         9       13       .44       3.29  7.40  12                                    18       13       .38       3.15  8.31   1                                    Control  17       .42       3.56  8.38  --                                    ______________________________________                                    

Kinashi et al. in Tetrahedron Lett. 49, 4955-4958 (1973) reported theantibiotic salinomycin to have the following structure (III): ##STR4##

As will be apparent upon comparison of salinomycin's structure with thatproposed for antibiotic A-28086 factor A, the structures are quitesimilar. A-28086 factor A differs only in that is has an additionalmethyl group on the E ring. A-28086 factors A, B, and D are, however,distinctly different from a comparison sample of salinomycin on variouspaper and thin-layer chromatographic systems.

This invention relates, therefore, to a method of increasingfeed-utilization efficiency in ruminants by administering apropionate-increasing amount of a compound selected from a groupconsisting of an A-28086 compound as specified, salinomycin, the C₂ -C₆-acyl ester derivatives of salinomycin, and thephysiologically-acceptable salts of salinomycin and of said esterderivatives. The physiologically-acceptable salts of salinomycin and ofits C₂ -C₆ -acyl ester derivatives are those salts as specified forA-28086 factor A and its C₂ -C₆ -acyl ester derivatives and are preparedin a similar manner. The C₂ -C₆ -acyl ester derivatives of salinomycinare prepared by the same method used to prepare the C₂ -C₆ -acyl esterderivatives of A-28086 factor A.

The compounds of this invention are typically effective in increasingpropionates and, thereby, the efficiency of feed-utilization whenadministered to ruminants orally at rates of from about 0.05 mg/kg/dayto about 5.0 mg/kg/day. Most beneficial results are achieved at rates offrom about 0.1 mg/kg/day to about 2.5 mg/kg/day. A preferred method ofadministration of a compound of this invention is by mixing it with theanimals' feed; however, it can be administered in other ways, forexample, tablets, drenches, boluses, or capsules. Formulation of thesevarious dosage forms can be accomplished by methods well known in theveterinary pharmaceutical art. Each individual dosage unit shouldcontain a quantity of a compound of this invention directly related tothe proper daily dose for the animal to be treated.

This invention further relates to feed compositions adapted to fattencattle comprising cattle feed and from 1 to 30 grams per ton of acompound selected from a group consisting of an A-28086 compound asspecified, salinomycin, the C₂ -C₆ -acyl ester derivatives ofsalinomycin, and the physiologically-acceptable salts of salinomycin andof said ester derivatives.

As is recognized by those skilled in the art, cattle feeds are differentfrom other feeds, such as poultry feeds. Cattle rations containroughages such as, for example, cotton-seed hulls and corn silage. Inaddition, cattle feeds contain a high percentage, often from 15-25percent, of non-protein nitrogen sources. A common non-protein nitrogensource is urea.

As described above, the A-28086 compounds are antiviral agents and arealso active against anaerobic bacteria, particularly Clostridiumperfringens. The A-28086 compounds are, therefore, beneficial in thetreatment or prevention of enteritis in chickens, swine, cattle andsheep. The A-28086 compounds are also useful in the treatment ofenterotoxemia in ruminants.

In order to illustrate more fully the operation of this invention thefollowing examples are provided.

EXAMPLE 1 A. Shake-flask Fermentation of A-28086 using S. aureofaciensNRRL 5758

A culture of Streptomyces aureofaciens NRRL 5758 was prepared andmaintained on an agar slant having the following composition:

    ______________________________________                                        Ingredient          Amount                                                    ______________________________________                                        Agar                20      g                                                 Dextrin             10      g                                                 Enzyme-hydrolyzed                                                              casein             2       g                                                 Beef extract        2       g                                                 Yeast extract       2       g                                                 Distilled water     q.s. 1  liter                                             ______________________________________                                    

The slant was inoculated with Streptomyces aureofaciens NRRL 5758, andthe inoculated slant was incubated at 30° C. for six to ten days. Themature slant culture was covered with beef serum, and scraped with asterile loop to loosen the spores. The resulting beef-serum suspensionof spores and mycelial fragments was lyophilized into six pellets.

One lyophilized pellet thus prepared was used to inoculate 50 ml of avegetative medium having the following composition:

    ______________________________________                                        Ingredient          Amount                                                    ______________________________________                                        Glucose             20      g                                                 Soybean grits       15      g                                                 Corn-steep liquor   10      g                                                 CaCO.sub.3          2       g                                                 Tap Water           q.s. 1  liter                                             ______________________________________                                    

The inoculated vegatative medium, in a 250-ml Erlenmeyer flask, wasincubated at 30° C. for 72 hours on a Shaker rotating through an arc 2inches in diameter at 250 rpm.

B. Tank Fermentation of A-28086 using S. aureofaciens NRRL 5758

In order to provide a larger volume of inoculum, 10 ml of the incubatedvegetative medium described above was used to inoculate 400 ml of asecond-stage vegetative growth medium having the same composition asthat of the vegetative medium. This second-stage medium, in a 2-literflask, was incubated at 30° C. for 24 hours on a shaker rotating throughan arc 2 inches in diameter at 250 rpm.

This second-stage vegetative medium (1 l.) was used to inoculate 100liters of sterile production medium of the following composition:

    ______________________________________                                        Ingredient            Amount                                                  ______________________________________                                        Tapioca dextrin       60.0    g/l.                                            Enzyme-hydrolyzed casein                                                                            8.0     g/l.                                            Molasses              15.0    g/l.                                            MgSO.sub.4 . 7H.sub.2 O                                                                             0.5     g/l.                                            CaCO.sub.3            2.0     g/l.                                            Refined soybean oil   5.0     g/l.                                            Deionized water       q.s. 1  liter                                           ______________________________________                                    

The pH of the medium was 6.7 after sterilization by autoclaving at 120°C. for 30 minutes at 15-20 pounds pressure. In a 165-liter fermentationtank, the inoculated production medium was allowed to ferment for 10days at a temperature of 29° C. The fermentation medium was aerated withsterile air at the rate of 0.4 volumes of air per volume of culturemedium per minute. The medium was stirred with conventional agitators at250 rpm.

EXAMPLE 2

The A-28086 antibiotics were produced according to the process ofExample 1, but utilizing a flask-production medium having the followingcomposition:

    ______________________________________                                        Ingredient            Amount                                                  ______________________________________                                        Glucose             10      g/l.                                              Edible molasses     20      g/l.                                              Peptone             5       g/l.                                              CaCO.sub.3          2       g/l.                                              Tap Water           q.s. 1  liter                                             ______________________________________                                    

EXAMPLE 3 Separation of the A-28086 Antibiotic Complex Produced by S.aureofaciens NRRL 5758

Whole fermentation broth (132 l.), obtained by the method described inExample 1, was filtered with a filter aid (Hyflo Super-cel, adiatomaceous earth, Johns-Manville Products Corp.) to give 97 liters offiltered broth. The filtered broth was extracted with an approximatelyequal volume of ethyl acetate. The ethyl acetate extract was separatedfrom the aqueous phase and was concentrated to a volume of about 500 ml.This concentrated ethyl acetate extract was added to a large excess ofpetroleum ether (Skelly Solve F; about 10 l.) to precipitate and,thereby, separate unwanted material. The separated filtrate wasevaporated under vacuum to give the broth portion of A-28086 antibioticcomplex (6.9 g).

The mycelial portion of A-28086 antibiotic complex was obtained byextracting the filtered mycelium twice with approximately half volumesof methanol (62 l. and 59 l.). The two methanol extracts were combinedand were concentrated under vacuum to remove the methanol. After thisconcentration about 10 l. of an aqueous phase remained. This aqueousphase was adjusted to about pH 7.5 with dilute sodium hydroxide. Theresulting solution was extracted twice with approximately equal volumesof ethyl acetate (9 l. and 10 l.). The ethyl acetate extracts werecombined and then concentrated to a volume of about 400 ml. Thisconcentrated ethyl acetate extract was added to a large excess ofpetroleum ether to remove unwanted materials, using the proceduredescribed above for the concentrated filtered broth extract. Themycelial portion of A-28086 antibiotic complex obtained from thefiltrate weighed 20.6 g.

EXAMPLE 4 Isolation of A-28086 Individual Factors A and B

The mycelial portion of A-28086 antibiotic complex (235 g, prepared asdescribed in Example 3) was dissolved in about 80 ml of benzene. Thisbenzene solution was applied to a silica gel column (9 × 130 cm, 8 l.,Matheson grade 62 silica gel). The column was eluted with varyingbenzene-ethyl acetate mixtures. Elution progress was followed bythin-layer chromatography. Using a benzene-ethyl acetate (90:10) solventsystem, factor B was eluted first and was isolated as an individualfactor. Factor B (43 mg) was crystallized from acetone-water, m.p.150°-153° C.

Continuing to elute with benzene-ethyl acetate mixtures but graduallyincreasing the ratio of ethyl acetate present, factor A was eluted; thevarious fractions containing factor A were combined and wereconcentrated under vacuum to a residue. This residue was dissolved inacetone (about 150 ml); water (about 150 ml) was added to the acetonesolution. The pH of the resulting solution was adjusted to pH 3 by theaddition of 1 N hydrochloric acid. The acidified mixture was stirredabout one hour, during which time a precipitate formed. This precipitatewas separated by filtration and was recrystallized from acetone (about150 ml) upon addition of water (about 60 ml). The product was driedovernight under vacuum to give factor A (about 6.6 g). After partialevaporation of acetone from the filtrate, a second crop of factor A(about 1.2 g) was obtained.

EXAMPLE 5 A-28086 Factor A-Acetyl Ester Derivative

Antibiotic A-28086 factor A (7.4 g) was dissolved in pyridine (150 ml).Acetic anhydride (50 ml) was added to this solution. The resultingsolution was mixed thoroughly and then was allowed to stand overnight atroom temperature.

Water (200 ml) was added, mixing thoroughly. This mixture was allowed tostand for four hours at room temperature. A white solid precipitated;this solid was separated by filtration, washed with water, and airdried. The resulting solid was dissolved in acetone (100 ml); and theacetone solution was evaporated to dryness under vacuum (this wasrepeated three times). The residue thus obtained crystallized fromacetone (100 ml)-water (50 ml), to give A-28086 factor A acetyl esterderivative (6.14 g), melting point 100°-103° C.

EXAMPLES 6-9

Antibiotic A-28086 factor A propionyl ester derivative, prepared byreacting factor A with propionic anhydride in the presence of pyridineaccording to the method of Example 5, melting point 96°-98° C.

Antibiotic A-28086 factor A n-butyryl ester derivative, prepared byreacting factor A with n-butyric anhydride in the presence of pyridineaccording to the method of Example 5, melting point 79°-81° C.

Antibiotic A-28086 factor A n-caproyl ester derivative, prepared byreacting factor A with caproic anhydride in the presence of pyridineaccording to the method of Example 5, melting point 163°-167° C.

Antibiotic A-28086 factor A n-valeryl ester derivative, prepared byreacting factor A with valeric anhydride in the presence of pyridineaccording to the method of Example 5, melting point 173°-175° C.

EXAMPLE 10 Preparation of A-28086 Factor A Sodium Salt

Antibiotic A-28086 factor A (500 mg) was dissolved in acetone (50 ml).Water (50 ml) was added to this solution, and 5 N sodium hydroxide wasadded to bring the pH of the solution to 10.5-11. The resulting solutionwas stirred for one hour and then was extracted with ethyl acetate. Theethyl acetate extract was evaporated to dryness under vacuum. Theresidue was precipitated from an acetone-water solution to give 378 mgof A-28086 factor A sodium salt, melting point 120°-123° C.

EXAMPLES 11-15

Antibiotic A-28086 factor A barium salt was prepared from antibioticA-28086 factor A (500 mg) and saturated barium hydroxide, using themethod of Example 10 to give 369 mg of A-28086 factor A barium salt,melting point 188°-190° C.

Antibiotic A-28086 factor A potassium salt was prepared from antibioticA-28086 factor A (500 mg) and 5 N potassium hydroxide, using the methodof Example 10 to give 363 mg of A-28086 factor A potassium salt, meltingpoint 165°-167° C.

Antibiotic A-28086 factor A cesium salt was prepared from antibioticA-28086 factor A (500 mg) and 1 N cesium hydroxide, using the method ofExample 10 to give 540 mg of A-28086 factor A cesium salt, melting point190°-210° C.

Antibiotic A-28086 factor B sodium salt, prepared from antibioticA-28086 factor B and 5 N sodium hydroxide according to the method ofExample 10.

EXAMPLE 16 Shake-flask Fermentation of A-28086 using S. aureofaciensNRRL 8092

A culture of Streptomyces aureofaciens NRRL 8092 was prepared andmaintained on an agar slant having the following composition:

    ______________________________________                                        Ingredient          Amount                                                    ______________________________________                                        K.sub.2 HPO.sub.4   2       g                                                 MgSO.sub.4 . 7H.sub.2 O                                                                           0.25    g                                                 NH.sub.4 NO.sub.3   2       g                                                 CaCO.sub.3          2.5     g                                                 FeSO.sub.4 . 7H.sub.2 O                                                                           0.001   g                                                 MnCl.sub.2 . 7H.sub.2 O                                                                           0.001   g                                                 ZnSO.sub.4 . 7H.sub.2 O                                                                           0.001   g                                                 Glucose             10      g                                                 Agar                20      g                                                 Deionized water-    q.s. 1  liter                                             pH (unadjusted)     7.7                                                       ______________________________________                                    

The slant was inoculated with Streptomyces aureofaciens NRRL 8092, andthe inoculated slant was incubated at 30° C. for about seven days. Themature slant culture was covered with sterile beef serum and was scrapedwith a sterile loop to prepare a spore and mycelial suspension from theslant culture. The resulting suspension was lyophilized into a maximumof six pellets.

One of the lyophile pellets thus prepared was used inoculate 50 ml of avegetative medium having the following composition:

    ______________________________________                                        Ingredient          Amount                                                    ______________________________________                                        Glucose             20      g                                                 Soybean flour       15      g                                                 Corn-steep liquor   10      g                                                 CaCO.sub.3          2       g                                                 Tap water           q.s. 1  liter                                             pH adjusted to pH 6.5 with dil NaOH                                           ______________________________________                                    

The inoculated vegetative medium, in a 250-ml Erlenmeyer flask, wasincubated at 30° C. for 48 hours on a rotary shaker at 250 rpm with atwo-inch arc.

The incubated vegetative medium described above (0.5 ml, 1 percent) wasused to inoculate 50 ml of a fermentation medium having the followingcomposition:

    ______________________________________                                        Ingredient              Amount                                                ______________________________________                                        Tapioca dextrin*        60.0    g                                             Enzyme-hydrolyzed casein**                                                                            6.0     g                                             Enzymatic hydrolysate of casein***                                                                    2.0     g                                             CaCO.sub.3              2.0     g                                             MgSO.sub.4 . 7H.sub.2 O 0.5     g                                             Blackstrap molasses     15.0    g                                             Refined soybean oil     5.0     ml                                            Tap water               q.s. 1  liter                                         pH (unadjusted) 6.6                                                           ______________________________________                                         *Staley Dextrin #11, A.E. Staley Co, Dacatur, Ill.                            **Amber EHC, Amber Laboratories, Juneau, Wics.                                ***NZ Amine A, Sheffield Chemical Co., Norwich, N.Y.                     

EXAMPLE 17 Tank Fermentation of A-28086 using S. aureofaciens NRRL 8092

The initial procedure described in Example 16 for the shake-flaskfermentation of A-28086 was also used for tank fermentation. In order toproduce a larger volume of inoculum, 10 ml of the incubated vegetativemedium was used to inoculate 400 ml of a second-stage vegetative mediumhaving the same composition as that of the first vegetative medium. Thissecond-stage medium, in a 2-liter Erlenmeyer flask, was incubated at 30°C. for 24 hours on a rotary shaker at 250 rpm with a two-inch arc.

This incubated second-stage vegetative medium (800 ml) was used toinoculate 100 liters of sterile fermentation medium having the followingcomposition:

    ______________________________________                                        Ingredient             Amount                                                 ______________________________________                                        Tapioca dextrin*       60.0    g/l.                                           Enzyme-hydrolyzed casein**                                                                           6.0     g/l.                                           Enzymatic-hydrolysate of casein***                                                                   2.0     g/l.                                           CaCO.sub.3             2.0     g/l.                                           MgSO.sub.4 . 7H.sub.2 O                                                                              0.5     g/l.                                           Blackstrap molasses    15.0    g/l.                                           Refined soybean oil    5.0     mg/l.                                          Tap water              q.s. 1  liter                                          ______________________________________                                         *Staley Dextrin, #11, A.E. Staley Co., Decatur, Ill.                          **Amber EHC, Amber Laboratories, Juneau, Wisc.                                ***NZ Amine A, Sheffield Chemical Co., Norwich, N.Y.                     

The pH of the medium was 6.8 ± 0.1 after sterilization by autoclaving at121° C. for 30 minutes at 15-20 pounds pressure. In a 165-literfermentation tank the inoculated production medium was allowed toferment for 10-12 days at 28° ± 1° C. The fermentation medium wasaerated with sterile air at the rate of 0.4 volumes of air per volume ofculture medium per minute. The medium was stirred with conventionalagitators at 300 rpm.

EXAMPLE 18 Separation of the A-28086 Antibiotic Complex Produced by S.aureofaciens NRRL 8092

Whole fermentation broth (60 liters), obtained by the method describedin Example 17, was adjusted to pH 3 by the addition of dilute HCl. Theresulting solution was filtered using a filter aid (Hyflo Super-cel, adiatomaceous earth, Johns-Manville Products Corp.). The separatedmycelial cake was extracted with 30 liters of methanol, adding 1.56 kgof NaHCO₃ to the extract with stirring. After separation of thisextract, the mycelial cake was again extracted with another 30 liters ofmethanol. The two methanol extracts were combined and concentrated undervacuum to remove the methanol. The remaining aqueous solution (about 7liters) was adjusted to pH 7.5 with dilute HCl. The resulting solutionwas extracted twice with ethyl acetate (7-liter portions). The ethylacetate extracts were combined and concentrated under vacuum to given anoily residue. This oily residue was dissolved in 1500 ml of acetone.Water (1500 ml) was added to the acetone solution. The resultingsolution was adjusted to pH 3 with dilute HCl and was stirred one hour.The precipitate which had formed was separated by filtration and thenwas dissolved in acetone (1500 ml); water (400 ml) was added to thissolution. The resulting solution was allowed to stand for 16 hours forcrystallization to occur. The crystals formed were separated byfiltration and dried under vacuum to give 74 g crude crystalline productcontaining A-28086 factors A and D and other crystalline impurities.

This crude crystalline product (40 g) was dissolved in about 250 ml ofbenzene. The benzene solution was then applied to a silica-gel column(9- × 120-cm column; Grace-Davidson grade 62 silica gal). The column waseluted successively with 40 liters of each of the following:

(1) benzene

(2) benzene:ethyl acetate (9:1)

(3) benzene:ethyl acetate (4:1)

(4) benzene:ethyl acetate (7:3)

(5) benzene:ethyl acetate (1:1)

(6) ethyl acetate

(7) methanol

One-liter fractions were collected. Each fraction was checked by assayagainst Bacillus Subtilis and by thin-layer chromatography to identifythe eluted compounds. A-28086I was eluted with benzene:ethyl acetate(4:1). A-28086 factor B was eluted with benzene:ethyl acetate (7:3).A-28086 factors A and D were eluted in the fractions obtained withbenzene:ethyl acetate (7:3 and 1:1), fractions 119-156. These fractionswere combined and evaporated to dryness under vacuum. The residue thusobtained was dissolved in acetone (500 ml). Water (500 ml) was added tothe acetone solution, and the resulting solution was adjusted to pH 3with dilute HCl and was stirred for one hour. The precipitate whichformed was separated by filtration and was crystallized from acetone(500 ml)-water (180 ml). The crystals thus formed were separated byfiltration and dried under vacuum to give 20.1 g of a mixture of A-28086factors A and D.

EXAMPLE 19 Separation and Purification of Individual Factors A and D

The crystalline mixture of A-28086 factors A and D obtained in Example18 (18.8 g) was dissolved in benzene (50 ml). The benzene solution wasapplied to a silica-gel column (7- × 100-cm column; E. Merck grade 60silica gel, finer than 230 mesh ASTM). The column was eluted, at a flowrate of 90 ml per hour, successively with:

(1) 12 liters of benzene

(2) 12 liters of benzene:ethyl acetate (9:1)

(3) 12 liters of benzene:ethyl acetate (4:1)

(4) 32 liters of benzene:ethyl acetate (7:3)

(5) 10 liters of methanol

Thin-layer cellulose chromatography (Merck Darmstadt cellulose onaluminum support) was followed by B. subtilis bioautography to monitorthe elution procedure. The following solvent system was used:water:methanol:acetone (12:3:1), adjusting the solution first to pH 10.5with NH₄ OH and then to pH 7.5 with HCl.

One- to two-liter fractions were collected until activity was detected;then 200-ml fractions were collected. The fractions containing onlyA-28086 factor D were combined and evaporated under vacuum to a residue.This residue crystallized from acetone-water (1:1). The crystals wereseparated and dried under vacuum to give 140 mg of crystalline A-28086factor D.

The fractions containing A-28086 factor D with a trace of A-28086 factorA were treated in the same manner to give an additional 150 mg ofcrystalline A-28086 factor D containing a small amount of A-28086 factorA.

The fractions containing only A-28086 factor A were also treated in thesame manner to give 4.7 g of crystalline A-28086 factor A.

EXAMPLE 20

The A-28086 antibiotics were produced according to the process ofExample 16, but using a slant medium having the following composition:

    ______________________________________                                        Ingredient             Amount                                                 ______________________________________                                        Beef extract           2.00     g                                             Dextrin                20.00    g                                             Yeast extract          2.00     g                                             Enzymatic-hydrolysate of casein                                                                      4.00     g                                             CoCl.sub.2 . 6H.sub.2 O                                                                              0.02     g                                             Agar                   20.00    g                                             Deionized water        q.s. 1   liter                                         pH adjusted to 7.0 with KOH                                                   ______________________________________                                    

and incubating the inoculated slant at 28° C. for about seven days.

EXAMPLE 21

The A-28086 antibiotics were produced according to the process ofExample 17, but using a flask/production medium having the followingcomposition:

    ______________________________________                                        Ingredient              Amount                                                ______________________________________                                        Tapioca dextrin*        80 g/l.                                               Enzyme-hydrolyzed casein**                                                                            15.0 g/l.                                             Blackstrap molasses     15.0 g/l.                                             Calcium carbonate       2.0 g/l.                                              Ammonium sulfate        1.0 g/l.                                              Magnesium sulfate       0.5 g/l.                                              Refined soybean oil     4.6 g/l.                                              ______________________________________                                         *Staley Dextrin #11, A.E. Staley Co., Decatur, Ill.                           **Amber EHC, Amber Laboratories, Juneau, Wisc.                           

EXAMPLE 22

The A-28086 antibiotics were produced according to the process ofExample 21, but using an intermediate third-stage vegetative mediumhaving the following composition:

    ______________________________________                                        Ingredient              Amount                                                ______________________________________                                        Cerelose                20.0 g/l.                                             Corn-steep liquor (wet wt.)                                                                           10.0 g/l.                                             Soybean grits           15.0 g/l.                                             CaCO.sub.3              2.0 g/l.                                              Yeast                   2.0 g/l.                                              Beet molasses           5.0 g/l.                                              ______________________________________                                    

EXAMPLE 23 A-28086-Modified Chick Ration for Coccidiosis Control

A balanced, high-energy ration adapted to feed chicks for rapid weightgain is prepared by the following recipe:

    ______________________________________                                        Ingredient            %      lbs                                              ______________________________________                                        Ground yellow corn    50     1,000                                            Soybean meal, solvent-                                                        extracted dehulled, finely                                                    ground, 50 percent protein                                                                          3109   621.8                                            Animal fat (beef tallow)                                                                            6.5    130                                              Dried fish meal, with                                                         solubles (60% protein)                                                                              5.0    100                                              Distiller's solubles                                                          from corn             4.0    80                                               Dicalcium phosphate, feed                                                     grade                 1.8    36                                               Calcium carbonate     0.8    16                                               Vitamin premix                                                                (representing vitamins A, D,                                                  E, K. and B.sub.12, choline, niacin,                                          pantothenic acid, riboflavin,                                                 biotin, with glucose bulking                                                  agent)                0.5    10                                               Trace mineral premix                                                          (representing MnSO.sub.4, ZnO,                                                KI, FeSO.sub.4, CaCO.sub.3)                                                                         0.2    4                                                2-Amino-4-hydroxybutyric acid                                                 (hydroxy analog of                                                            methionine)           0.1    2                                                A-28086 Factor A      0.01   0.2                                              ______________________________________                                    

These substances are mixed in accordance with standard feed-mixingtechniques. Chicks fed such a ration, with water ad libitum, areprotected against exposure to coccidiosis; weight gains are comparableto those of coccidiosis-free chicks fed a similar, unmedicated diet.

EXAMPLE 24 A-28086-Improved Beef-Cattle Ration

A balanced high-grain beef-cattle ration is prepared as follows:

    ______________________________________                                        Ingredient          %        lbs                                              ______________________________________                                        Finely ground corn  67.8     1356                                             Ground corn cob     10       200                                              Dehydrated alfalfa meal,                                                      17 percent protein  5        100                                              Dehulled soybean meal,                                                        solvent extracted,                                                            50 percent protein  9.9956   199.912                                          Cane molasses       5        100.0                                            Urea                0.6      12.0                                             A-28086 Factor A    0.0044   0.088                                            Dicalcium phosphate,                                                          feed grade          0.5      10.0                                             Calcium carbonate   0.5      10.0                                             Sodium chloride     0.3      6.0                                              Trace mineral premix                                                                              0.03     0.6                                              Vitamin A and D.sub.2 premix*                                                                     0.07     1.4                                              Vitamin E premix**  0.05     1.0                                              Calcium propionate  0.15     3.0                                              ______________________________________                                         *Containing per pound: 2,000,000 I.U. of vitamin A; 227,200 I.U. of           vitamin D.sub.2 and 385.7 g of soybean feed with 1% oil added.                **Corn distillers dried grains with solubles containing 20,000 I.U. of        d-alpha-tocopheryl acetate per pound                                     

The mixed feed is compressed into pellets. At an average daily ingestionrate of 15 pounds of feed per animal, this feed supplies approximately300 mg of A-28086 factor A per animal per day.

We claim:
 1. A method of increasing feed-utilization efficiency in aruminant animal which comprises orally administering to said animal inneed of increasing the feed-utilization efficiency an effectivepropionate-increasing amount of salinomycin or aphysiologically-acceptable salt of salinomycin.
 2. The method of claim 1wherein the compound is salinomycin.